Epithelial-to-mesenchymal transition (EMT) has emerged as a critical step in the early stage of cancer. Better understanding of the mechanism of EMT will foster the development of inhibitors to prevent cancer progression. Considerable evidence has implicated membrane type 1-matrix metalloproteinase (MT1-MMP) in EMT and conversion to aggressive cancers. MT1-MMP activation of proMMP-2 and degradation of extracellular matrix (ECM) components are involved in cancer progression. MT1-MMP also cleaves numerous cell surface proteins, growth factors, and chemokines. We recently demonstrated that MT1-MMP: 1) cleaves E-cadherin at the cell-cell adherins junctions;2) promotes cell migration/scattering in 3D type I collagen gels; 3) regulates cell proliferation in vitro and in vivo;and 5) changes cell morphology in 3D type I collagen gels. We also demonstrated that the hemopexin (PEX) domain of MT1-MMP plays a crucial role in cancer cell migration and pinpointed the regions required for MT1-MMP-mediated cell migration. Targeting the PEX domain with recombinant MT1-MMP PEX protein resulted in interference with MT1-MMP-induced cell migration. The primary goal of this grant is to define the involvement of the catalytic and non catalytic activities of MT1 -MMP in EMT. This characterizationwill facilitate our development of specific non-catalytic domain inhibitors of MT1-MMP that will be employed to interfere with cancer progression. To achieve this aim, the function of MT1-MMP in EMT in 3D cultured cancer cells will be examined. EMT-related transcription programs and signaling pathways will be evaluated. Based on a computational model of MT1-MMP and similarity with other MMPs, minimum motif(s) in the PEX domain of MT1-MMP required for cancer cell migration will be identified using a mutagenesis approach. Formation of homodimer and/or heterooligomer of MT1-MMP through PEX domain will be determined. Based on the identification of crucial PEX motifs, specific MT1-MMP inhibitory peptides will be designed and characterized using analytical chemistry and cell biological approaches. Functional inhibitory peptides will be produced and evaluated in an in vivo cancer model. The long-term goal is to develop a lead compound that will be modified to produce a MT1-MMP inhibitory drug with minimal side effects for treatment of cancer. I propose that the current project will help us to better understand cancer progression and lead to the development of novel basic tools for treatment of early stage cancer.